Melatonin attenuates calpain upregulation, axonal damage and neuronal death in spinal cord injury in rats

Abstract

Multiple investigations in vivo have shown that melatonin (MEL) has a neuroprotective effect in the treatment of spinal cord injury (SCI). This study investigates the role of MEL as an intervening agent for ameliorating Ca(2+)-mediated events, including activation of calpain, following its administration to rats sustaining experimental SCI. Calpain, a Ca(2+)-dependent neutral protease, is known to be involved in the pathogenesis of SCI. Rats were injured using a standard weight-drop method that induced a moderately severe injury (40 g.cm force) at T10. Sham controls received laminectomy only. Injured animals were given either 45 mg/kg MEL or vehicle at 15 min post-injury by intraperitoneal injection. At 48 hr post-injury, spinal cord (SC) samples were collected. Immunofluorescent labelings were used to identify calpain expression in specific cell types, such as neurons, glia, or macrophages. Combination of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and double immunofluorescent labelings was used to identify apoptosis in specific cells in the SC. The effect of MEL on axonal damage was also investigated using antibody specific for dephosphorylated neurofilament protein (dNFP). Treatment of SCI animals with MEL attenuated calpain expression, inflammation, axonal damage (dNFP), and neuronal death, indicating that MEL provided neuroprotective effect in SCI. Further, expression and activity of calpain and caspse-3 were examined by Western blotting. The results indicated a significant decrease in expression and activity of calpain and caspse-3 in SCI animals after treatment with MEL. Taken together, this study strongly suggested that MEL could be an effective neuroprotective agent for treatment of SCI.

Fig. 1

The effects of melatonin (MEL) on expression and activity of calpain and caspase-3 following acute spinal cord (SC) injury. (A) Representative Western blots using lesion and caudal penumbra SC segments from sham and SCI rats treated with vehicle (dimethyl sulfoxide) or MEL (45 mg/kg) for 48 hr. (B) Determination of OD of bands representing calpain expression and activity. (C) Determination of OD of bands representing caspase-3 active fragment and activity. Data presented as percentage change in comparison with sham + vehicle set at 100% (n = 3). Sham + vehicle vs. injury + vehicle (*P ≤ 0.05); and injury + vehicle vs. injury + MEL (^@P ≤ 0.05).

Fig. 2

The effects of melatonin (MEL) on calpain expression in neurons following acute spinal cord (SC) injury. Double immunofluorescent labelings were performed using lesion and caudal penumbra SC sections from sham and SCI rats treated with vehicle or MEL (45 mg/kg) for 48 hr. Calpain expression in neurons was assessed using calpain antibody (red) and NeuN antibody (green). Arrows show co-staining (n = 3, 200× magnification).

Fig. 3

The effects of melatonin (MEL) on neuronal death following acute spinal cord (SC) injury. (A) Double immunofluorescent labelings for detecting apoptotic death in neurons were performed using lesion and caudal penumbra SC sections from sham and SCI rats treated with vehicle or MEL (45 mg/kg) for 48 hr. Apoptotic death in neurons was assessed by TUNEL staining (red) and NeuN staining (green). Arrows show co-staining (n = 3, 200× magnification). (B) Quantification of TUNEL-positive cells. Sham + vehicle vs. injury + vehicle (*P ≤ 0.05); and injury + vehicle vs. injury + MEL (^#P < 0.05).

Fig. 4

The effects of melatonin (MEL) on infiltration of inflammatory cells following acute spinal cord (SC) injury. Double immunofluorescent labelings of macrophages/microglia (inflammatory cells) and calpain expression were performed using lesion and caudal penumbra SC sections from sham and SCI rats treated with vehicle or MEL (45 mg/kg) for 48 hr. Macrophages/microglia and calpain expression were assessed using OX42 antibody (green) and calpain antibody (red), respectively. Arrows show co-staining (n = 3, 200× magnification).

Fig. 5

The effects of melatonin (MEL) on astrogliosis following acute spinal cord (SC) injury. Double immunofluoresecent labelings for astrogliosis and calpain expression were performed using lesion and caudal penumbra SC sections from sham and SCI rats treated with vehicle or MEL (45 mg/kg) for 48 hr. Astrogliosis and calpain expression were assessed using GFAP antibody (green) and calpain antibody (red), respectively. Arrows depict astrogliosis. Arrowheads show co-staining (n = 3, 200× magnification).

Fig. 6

The effects of melatonin (MEL) on axonal damage following acute spinal cord (SC) injury. (A) Single immunofluorescent labeling for axonal damage was performed using lesion and caudal penumbra SC sections from sham and SCI rats treated with vehicle or MEL (45 mg/kg) for 48 hr. Use of dephosphorylated neurofilament protein (dNFP) antibody (red) detected axonal damage (n = 3, 200× magnification). (B) Determination of dNFP. Data were analyzed with NIH ImageJ software. Significant differences in the number of pixels were indicated: sham + vehicle vs. injury + vehicle (*P ≤ 0.05); and injury + vehicle vs. injury + MEL (^#P < 0.05).